Bacterial Protein E.P.C (Expression, Purification and Characterization)
Introduction:
The use of recombinant proteins can be useful in many areas of microbiology, from epidemiology to virology and onwards [1]. This lab consists of three parts: expression, purification, and characterization. The purpose of the first part, expression, was to overexpress recombinant protein in bacteria. We transformed bacterial cells with a DNA plasmid and grew a starter culture of bacteria, and then used this culture to harvest a larger culture to express the recombinant protein. The purpose of the purification section was to isolate the protein gbr-22, getting rid of excess protein and waste. The final step of the lab, characterization, was done to electrophoresis the samples from the first two labs and analyze them. Materials & Methods:
In the expression portion, we added 25uL of the bacteria, which was BL21(DE3), was placed in a transformation tube. Then, 2 uL of the plasmid, pGEM-gbr22 (gbr stands for Great Barrier Reef), was added to the tube. The tube, containing both the bacteria and the plasmid, must then be placed on ice for 30 minutes. After this period of time, we heat shock the tubes in 42-degree (Celsius) water for 45 seconds. After this portion, 200uL of SOC media must be added to the tube. This then needs to be placed in an incubator. Then 50 uL of the bacteria medium must be pipetted onto a gel plate, for growth. We then left the plates overnight to allow the bacteria culture to grow. In day 2 of the expression portion, we added ampicillin to a tube of 5 ml LB. We then swipe a portion of the bacteria culture onto a pipette tip, and place it in the tube of LB and ampicillin. We then transfer 25 ml of LB to an Erlenmeyer flask, and add ampicillin again. We then add .625 ml of the starter culture to the flask. After this, we place the flask in a shaking incubator for 16-24 hours. We then use the large bench-top centrifuge to harvest the cells. Before this, take a 500 uL sample of the culture and place it in an Eppendorf tube. We then placed the bacterial in a conical tube and centrifuged it for 10 minutes at 5000 rpm and 4-degrees Celsius. We then decant the liquid out and save the pellet. The next step was purification. The first step was to filter the lysate with a syringe. Then make the resin/buffer mix (Ni-NTA added to culture). After this, the mixture must be incubated. Following this, a BioRad chromatography column must be set up. This step is the actual purification portion. Add the resin to the column, and once the resin settles, let the waste flow out from the column. Next, begin the wash step. This is to remove loosely bound proteins. This is done my adding imidazole to the column. The gbr-22 protein is released through the elution step. This requires a high concentration of imidazole. The elution sample will be purplish. In part 3 of this lab, we used samples taken from part 2. A loading buffer must be acquired, which is 6x concentration. Sample 1 collected from the Purification lab must be centrifuged and resuspended. Samples 2-6, which consisted of the wash and elution samples, had to be heat shocked at 95 degrees for 5 minutes. Afterwards, the electrophoresis module had to be assembled, and the protein added to the docks along with a DNA ladder. This electrophoresis took about 25 minutes. Following the electrophoresis, the gel had to be placed in a containers, rinsed, and placed on an orbital shaker for 1.5 hours. After this, the gel must be rinsed again, and placed on the shaker repeatedly at 5 minute intervals. Finally, the gel is placed on the orbital shaker with a paper towel overnight. The next day, the gel is dried.
Results:
Figure 1. Ecoli culture. The bacteria with the protein gbr-22 are ampicillin resistant, and will have a purplish color.
Figure 2. Control plate has no growth because bacteria are not ampicillin resistant
Figure 3. Ecoli that expresses the gbr-22, mixed with LB and ampicillin
Figure 4. Concentrated pellet of the protein, supernatant was decanted out
Figure 5. Elution 1, containing most of the purified protein
Figure 6. Elution 2, gathering any excess protein
Figure 7: Nanodrop spectrophotometer absorbance spectra for Elution 1 sample at 280 nanometers
Figure 8. Second run of Elution 1 at 280 nanometers.
Figure 9. Nanodrop for Elution 1 at 574 (max) nanometers.
Figure 10. Run 2 for Elution 1 at 574 nanometers
Figure 11. Gel electrophoresis at 200 volts for 25 minutes. The gel was then stained and dried. The top row was the DNA ladder, followed by sample 1-6 and 4-6. Sample 5 had the strongest concentration of the protein.
Figure 12. Standard molecular weight for electrophoresis
Discussion: Lysozyme is used because it breaks down the cell walls of gram negative bacteria, such as E.coli. Gram negative bacteria have less peptidoglycan in their cell walls than do gram positive bacteria. These gram negative bacteria have more complex walls, and are thus harder to eradicate. Lysozyme is strong and can break down these gram negative bacterial walls. Benzoase gets rid of excess cell debris. Sample 1 contains the bacteria in question. Sample 2 contains the lysate added supernatant. Sample 3 contains the waste from the column step. Sample 4 contained the wash, which was achieved by adding imidazole and removing loosely bound proteins. Sample 5 and 6 contained the two elution samples. The wash buffer contained imidazole to get rid of the excess proteins that are not being examined. The elution buffer is added to remove the gbr-22, the protein being examined, and it does this because it contains a higher concentration of imidazole. The way imidazole works is it mimics the Histidine and binds where histidine normally would, so the actual protein doesn't bind or binds loosely, and thus washes off.
Conclusions:
This lab demonstrated how to isolate a protein by using recombinant DNA and making a certain bacteria, in this case E.coli, produce a certain protein. This is done through 3 steps, expression, purification, and characterization. After gel electrophoresis, many properties of the protein can be determined, such as the molecular weight. Future uses of this procedure are to isolate certain proteins of harmful viruses and bacteria and examine them, perhaps preventing an epidemic from outbreaking.
References: [1] Wang R, Wang J, Li J, Wang Y, Xie Z, An L: Comparison of two gel filtration chromatographic methods for the purification of Lily symptomless virus. Journal of Virological Methods 2007, 139:125-131.
Introduction:
The use of recombinant proteins can be useful in many areas of microbiology, from epidemiology to virology and onwards [1]. This lab consists of three parts: expression, purification, and characterization. The purpose of the first part, expression, was to overexpress recombinant protein in bacteria. We transformed bacterial cells with a DNA plasmid and grew a starter culture of bacteria, and then used this culture to harvest a larger culture to express the recombinant protein. The purpose of the purification section was to isolate the protein gbr-22, getting rid of excess protein and waste. The final step of the lab, characterization, was done to electrophoresis the samples from the first two labs and analyze them.
Materials & Methods:
In the expression portion, we added 25uL of the bacteria, which was BL21(DE3), was placed in a transformation tube. Then, 2 uL of the plasmid, pGEM-gbr22 (gbr stands for Great Barrier Reef), was added to the tube. The tube, containing both the bacteria and the plasmid, must then be placed on ice for 30 minutes. After this period of time, we heat shock the tubes in 42-degree (Celsius) water for 45 seconds. After this portion, 200uL of SOC media must be added to the tube. This then needs to be placed in an incubator. Then 50 uL of the bacteria medium must be pipetted onto a gel plate, for growth. We then left the plates overnight to allow the bacteria culture to grow. In day 2 of the expression portion, we added ampicillin to a tube of 5 ml LB. We then swipe a portion of the bacteria culture onto a pipette tip, and place it in the tube of LB and ampicillin. We then transfer 25 ml of LB to an Erlenmeyer flask, and add ampicillin again. We then add .625 ml of the starter culture to the flask. After this, we place the flask in a shaking incubator for 16-24 hours. We then use the large bench-top centrifuge to harvest the cells. Before this, take a 500 uL sample of the culture and place it in an Eppendorf tube. We then placed the bacterial in a conical tube and centrifuged it for 10 minutes at 5000 rpm and 4-degrees Celsius. We then decant the liquid out and save the pellet. The next step was purification. The first step was to filter the lysate with a syringe. Then make the resin/buffer mix (Ni-NTA added to culture). After this, the mixture must be incubated. Following this, a BioRad chromatography column must be set up. This step is the actual purification portion. Add the resin to the column, and once the resin settles, let the waste flow out from the column. Next, begin the wash step. This is to remove loosely bound proteins. This is done my adding imidazole to the column. The gbr-22 protein is released through the elution step. This requires a high concentration of imidazole. The elution sample will be purplish. In part 3 of this lab, we used samples taken from part 2. A loading buffer must be acquired, which is 6x concentration. Sample 1 collected from the Purification lab must be centrifuged and resuspended. Samples 2-6, which consisted of the wash and elution samples, had to be heat shocked at 95 degrees for 5 minutes. Afterwards, the electrophoresis module had to be assembled, and the protein added to the docks along with a DNA ladder. This electrophoresis took about 25 minutes. Following the electrophoresis, the gel had to be placed in a containers, rinsed, and placed on an orbital shaker for 1.5 hours. After this, the gel must be rinsed again, and placed on the shaker repeatedly at 5 minute intervals. Finally, the gel is placed on the orbital shaker with a paper towel overnight. The next day, the gel is dried.
Results:
Figure 1. Ecoli culture. The bacteria with the protein gbr-22 are ampicillin resistant, and will have a purplish color.
Figure 2. Control plate has no growth because bacteria are not ampicillin resistant
Figure 3. Ecoli that expresses the gbr-22, mixed with LB and ampicillin
Figure 4. Concentrated pellet of the protein, supernatant was decanted out
Figure 5. Elution 1, containing most of the purified protein
Figure 6. Elution 2, gathering any excess protein
Figure 7:
Nanodrop spectrophotometer absorbance spectra for Elution 1 sample at 280 nanometers
Figure 8. Second run of Elution 1 at 280 nanometers.
Figure 9. Nanodrop for Elution 1 at 574 (max) nanometers.
Figure 10. Run 2 for Elution 1 at 574 nanometers
Figure 11. Gel electrophoresis at 200 volts for 25 minutes. The gel was then stained and dried. The top row was the DNA ladder, followed by sample 1-6 and 4-6. Sample 5 had the strongest concentration of the protein.
Figure 12. Standard molecular weight for electrophoresis
Discussion:
Lysozyme is used because it breaks down the cell walls of gram negative bacteria, such as E.coli. Gram negative bacteria have less peptidoglycan in their cell walls than do gram positive bacteria. These gram negative bacteria have more complex walls, and are thus harder to eradicate. Lysozyme is strong and can break down these gram negative bacterial walls. Benzoase gets rid of excess cell debris. Sample 1 contains the bacteria in question. Sample 2 contains the lysate added supernatant. Sample 3 contains the waste from the column step. Sample 4 contained the wash, which was achieved by adding imidazole and removing loosely bound proteins. Sample 5 and 6 contained the two elution samples. The wash buffer contained imidazole to get rid of the excess proteins that are not being examined. The elution buffer is added to remove the gbr-22, the protein being examined, and it does this because it contains a higher concentration of imidazole. The way imidazole works is it mimics the Histidine and binds where histidine normally would, so the actual protein doesn't bind or binds loosely, and thus washes off.
Conclusions:
This lab demonstrated how to isolate a protein by using recombinant DNA and making a certain bacteria, in this case E.coli, produce a certain protein. This is done through 3 steps, expression, purification, and characterization. After gel electrophoresis, many properties of the protein can be determined, such as the molecular weight. Future uses of this procedure are to isolate certain proteins of harmful viruses and bacteria and examine them, perhaps preventing an epidemic from outbreaking.
References:
[1] Wang R, Wang J, Li J, Wang Y, Xie Z, An L: Comparison of two gel filtration chromatographic methods for the purification of Lily symptomless virus.
Journal of Virological Methods 2007, 139:125-131.